Field of the Invention
The present invention relates to analysis of a trace amount of nucleic acid component such as RNA or DNA contained in one or a few cells, and particularly relates to a method of producing cDNA from mRNA and equally amplifying cDNA.
Background Art
Gene expression analysis has been widely used as a means for accurately elucidating the state of a living body. As a typical expression analysis method, a real-time PCR method and a micro-array method have been known. For the analysis, mRNA is extracted from various samples and put in use. For example, mRNA extracted from cultured and proliferated cells or mRNA extracted from a tissue piece containing a plurality of cells has been frequently used. An amount of each mRNA measured is regarded as an average amount of mRNA molecules collected from a plurality of cells and to be subjected to analysis.
In the meantime, a significant development has been recently made in the academic field. In a tissue piece and a cell population, which are up to present regarded as a group of homogeneous cells and collected, it has been found that a plurality types of cells are present. For example, a cancer tissue contains a cancer stem cell, from which cancer is originated. It has been elucidated that cancer cells are supplied from the cancer stem cell. Furthermore, in embryonic stem (ES) cells application of which to regenerative medicine has been expected, it has been elucidated that individual cells of the ES cell population are not homogeneous and that cells having a different differentiation potency are present in the population. In the studies dealing with cells whose characteristics differ from each other, a conventional method which analyzes an expression based on the average expression of a cell population is no longer sufficient. There is a possibility that true expression analysis could not be made unless genes of individual cells are analyzed at the single-cell level.
However, the amount of mRNA contained in a single cell is extremely small. The amount of mRNA contained in a cultured human cell is presumably about 2 pg or less. It is difficult to analyze expression of a trace amount of mRNA by a conventional method. In the circumstances, to realize the expression analysis of a trace amount of mRNA derived from a single cell, various approaches have been developed.
As a method for a quantitative gene expression analysis, a real-time PCR method is excellent. The present inventors have already reported on the method for analyzing the amount of mRNA derived from a single cell by a real-time PCR method. More specifically, in the method we developed, mRNA is extracted from a single cell and a cDNA library is constructed on magnetic beads and used as a sample for real time PCR (Patent Document 1 and Non Patent Document 1). In the method, amplification is performed by use of different real-time PCR primers between genes and compared to a calibration curve of a standard sample amplified by the same primer. In this manner, accurate quantitative analysis can be realized. In this method, however, since the number of genes simultaneously measured is limited to several, it was difficult to make comprehensive expression analysis.
In contrast, as a method for a comprehensive gene expression analysis, a micro-array method is excellent. Kurimoto et al., have realized comprehensive expression analysis of a single cell in combination with a global amplification method (Patent Document 2, Non Patent Documents 2 and 3). However, the micro-array method may be inferior in quantitative performance, since the intensity of expression is measured based on hybridization of a gene-sequence specific probe compartmentally arranged on a chip, with a sample nucleic acid.
The real-time PCR method and the micro-array method have a following common problem: a sample the nucleotide sequence of which has not been determined, cannot be analyzed. In a cell, various unknown variant mRNA molecules and many unknown non-coding RNA molecules involved in gene expression regulation although they do not encode proteins, are present. Importance of expression analysis of these unknown RNA molecules in elucidating life has been extremely increased in recent years.
Under such circumstances, large-scale DNA sequencers have been recently developed. With this development, a novel method, that is, an expression analysis method (RNA-Seq), has been attracted attention, in which the sequence analysis of mRNA is made based on the analysis of cDNA produced from the mRNA by a DNA sequencer. RNA-Seq is capable of simultaneously analyzing nucleotide sequences of tens of millions to several hundred-millions of DNA fragments. The sequences thus analyzed are subjected to a mapping operation using a known gene sequence, with a computer. The number of fragments whose sequences are mapped is counted to computationally obtain the expression amount of RNA.
In RNA-Seq, expression of unknown mRNA can be analyzed as long as a reference genomic DNA sequence for use in mapping is known. Thus, RNA-Seq has a great advantage in overcoming drawbacks of conventional expression analysis methods. Furthermore, the dynamic range of measurement is as wide as 5 log. Thus, it is considered that all mRNA molecules expressed in a cell can be quantitatively measured at a time. Moreover, in RNA-Seq, expression amounts of genes are not simply compared but the sequences thereof can be analyzed, with the result that information, such as a mutation of gene itself, can be obtained other than that obtained by quantitative analysis. Therefore, RNA-Seq has a large impact on the field of bioscience as an innovative analysis method.
As described above, RNA-Seq by a large-scale DNA sequencer is an extremely excellent analysis method; however, assuming that expression of a single cell is analyzed by the sequencer, there is a large problem that is to be overcome. This is a process of amplifying DNA, which is essential since a large-scale sequencer requires a large amount of DNA as an analysis sample. For example, in preparing samples for a SOLiD sequencer (Lifetech Co., Ltd.), a step of individual amplification by emulsion PCR is required. Before the step, it is necessary to amplify a DNA sample up to approximately several hundreds of ng. In this amplification process, several steps are required for preparing a sample so as to satisfy the specifications defined by a sequencer, such as addition of an amplification adaptor and size selection based on DNA fragment length. Because of this, it is very difficult to actually amplify DNA up to several hundreds of ng so as to satisfy the specifications defined by a large-scale sequencer based on a trace amount of mRNA derived from a single cell. Even if amplification can be made, amplification does not equally proceed during amplification. As a result, there is a high possibility that the obtained sample fails to reflect the expression amount ratio of mRNA molecules in a single cell.
Kits for preparing a sample for RNA-Seq so as to satisfy the specifications defined by individual sequencers are available from various manufacturers; however, none of the kits fail to prepare a sample from a single cell-level mRNA of 2 pg or less. Accordingly, in order to perform RNA-Seq of a single cell, it is necessary to develop an original method for preparing a sample. Such a method has been reported in some papers.
For example, Tang et al. improved the aforementioned global amplification method and succeeded in expression analysis in single-cell such as an ovum or an ES cell of a mouse by using a large-scale sequencer (Non Patent Documents 4 and 5). The global amplification method includes the following steps:
(1) extracting mRNA from a single cell and synthesizing cDNA by use of DNA primer (UP1) having a poly T sequence and a tag sequence for PCR amplification; (2) degrading the remaining unreacted DNA primer in cDNA synthesis, with single-strand DNA degradation enzyme, i.e., exonuclease I; (3) inactivating exonuclease I with heat, and then degrading mRNA with RNaseH and adding a poly A sequence to the 3′ terminal of cDNA with TdT (Terminal Deoxynucleotidyl Transferase); (4) performing synthesis reaction of a double-strand by use of a DNA primer (UP2) having a tag sequence for amplification, which is different from that of the UP1 primer as mentioned above, and a poly T sequence; and (5) performing PCR amplification using the double-stranded DNA synthesized as a template, with a set of UP1 primer and UP2 primer.
This method is suitable for amplification of a trace amount of sample. Generally, if such a method is used, a serious problem, unequal amplification, may be accompanied. Unequal amplification refers to a phenomenon where the amplification rates of individual genes vary in a step of amplifying the genes, as previously described, with the result that the obtained results do not reflect the expression amount ratio of intracellular mRNA molecules. In other words, this is referred to as an amplification bias, which is going to be a significant problem in quantitatively comparing gene expression amounts.
The gene expression amount found in elucidating development and differentiation greatly varies depending upon the stage. Thus, if several-fold amplification bias is present, no significant problem occurs in some cases. However, if a marker is searched in the field of medicine where a further development is expected, there is a case where genes are desired to be distinguished even if the expression amounts of them differ only about twofold. In such a case, it is difficult to distinguish such small difference in expression amount between the genes by the expression analysis method giving a several-fold amplification bias. Because of this, it has been desired to develop a method directed to a single-cell mRNA for preparing a sufficient amount of sample for a large-scale sequencer and providing less amplification bias as much as possible.